Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation

Abstract

The present invention is directed to a polymeric carrier cargo complex, comprising as a cargo at least one nucleic acid (molecule) and disulfide-crosslinked cationic components as a (preferably non-toxic and non-immunogenic) polymeric carrier. The inventive polymeric carrier cargo complex allows for both efficient transfection of nucleic acids into cells in vivo and in vitro and/or for induction of an (innate and/or adaptive) immune response, preferably dependent on the nucleic acid to be transported as a cargo. The present invention also provides, pharmaceutical compositions, particularly vaccines and adjuvants, comprising the inventive polymeric carrier cargo complex and optionally an antigen, as well as the use of such the inventive polymeric carrier cargo complex and optionally an antigen for transfecting a cell, a tissue or an organism, for (gene-)therapeutic purposes as disclosed herein, and/or as an immunostimulating agent or adjuvant, e.g. for eliciting an immune response for the treatment or prophylaxis of diseases as mentioned above. Finally, the invention relates to kits containing the inventive polymeric carrier cargo complex and/or the inventive pharmaceutical composition, adjuvant or vaccine in one or more parts of the kit.

1 . A polymeric carrier cargo complex, comprising:
 a) as a carrier a polymeric carrier formed by disulfide-crosslinked cationic components, and   as a cargo at least one nucleic acid molecule.   
     
     
         2 . The polymeric carrier cargo complex according to  claim 1 , wherein the at least one nucleic acid molecule is an RNA. 
     
     
         3 . The polymeric carrier cargo complex according to  claim 2 , wherein the at least on nucleic acid molecule is an immunostimulatory nucleic acid. 
     
     
         4 . The polymeric carrier cargo complex according to  claim 2 , wherein the at least on nucleic acid molecule is an immunostimulatory RNA (isRNA) or an mRNA. 
     
     
         5 . A polymeric carrier cargo complex according to  claim 2 , wherein the nitrogen/phosphate (N/P) ratio of the cationic components to the at least one nucleic acid molecule is in the range of 0.1-20, or in the range of 0.1-5, or in the range of 0.1-1. 
     
     
         6 . A polymeric carrier cargo complex according to  claim 1 , wherein the polymeric carrier further comprises functional peptides or proteins. 
     
     
         7 . A polymeric carrier cargo complex according to  claim 6 , wherein the functional peptides or proteins are peptide or protein antigens or antigen epitopes. 
     
     
         8 . A polymeric carrier cargo complex according to  claim 2 , wherein the polymeric carrier further comprises a ligand. 
     
     
         9 . A polymeric carrier cargo complex according to  claim 8 , wherein the ligand is mannose. 
     
     
         10 . The polymeric carrier cargo complex according to  claim 2 , wherein the cationic components are cationic peptides. 
     
     
         11 . The polymeric carrier cargo complex according to  claim 10 , wherein the cationic peptides
 are selected from peptides according to formula (I)
   (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x , 
   wherein   l+m+n+o+x=3-100, and   l, m, n or o=independently of each other is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90 and 91-100, provided that the overall content of Arg, Lys, His and Orn represents at least 10% of all amino acids of the cationic peptide; and Xaa is any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and   x=any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, and 91-100 provided that the overall content of Xaa does not exceed 90% of all amino acids of the cationic peptide, or are selected from peptides according to subformula (Ia)
   {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa′) x (Cys) y }
 
   or from peptides according to subformula (Ib)
   Cys 1 {(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x }Cys 2    
   wherein (Arg) l ; (Lys) m ; (His) n ; (Orn) o ; and x are as defined above; Xaa′ is any amino acid selected from native or non-native amino acids except of Arg, Lys, His, Orn; or Cys and y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, and 91-100 provided that the overall content of Arg (Arginine), Lys (Lys 1  ne), His (Histidine) and Orn (Ornithine) represents at least 10% of all amino acids of the oligopeptide and wherein Cys 1  and Cys 2  are Cysteines proximal to, or terminal to (Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x .   
     
     
         12 . The polymeric carrier cargo complex according to  claim 1 , wherein the disulfide-bonds are formed by cysteine residues contained in the cationic peptides. 
     
     
         13 . A polymeric carrier cargo complex according to  claim 12 , wherein the cysteine residue is located at the terminal ends of the cationic peptides. 
     
     
         14 . A method of stimulating an immune response in a subject comprising administering to the subject a polymeric carrier cargo complex according to  claim 1 . 
     
     
         15 . A vaccine comprising a polymeric carrier cargo complex as defined according to  claim 1  and an antigen or RNA encoding an antigen.